Findings from FY10 are summarized below. 1. Animal models of orthopoxviruses and countermeasures. Cases of monkeypox virus (MPXV) infection in humans are expanding in Africa and can be fatal in up to 10% of affected individuals. In addition, the virus was imported into the United States in 2003 and caused a localized outbreak in the Midwest. Based on the growing threat of MPXV disease and concerns that variola might be used as an agent of bioterrorism, we have continued to focus on the study of orthopoxvirus pathogenesis in animals and the identification of countermeasures. We are exploring alternate routes of MPXV inoculation in nonhuman primates (NHP) to identify a model of infection that most closely resembles natural monkeypox and variola disease progression in humans. The commonly utilized intravenous (IV) route of infection results in an accelerated fulminant disease course compared to MPXV infection in humans. Our goal was to determine if intrabronchial (IB) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. We have compared the disease course following IV or IB inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the IB model when compared to the IV model including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by inoculation route at the highest doses tested. The NHPs inoculated by the IB route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble human MPXV infection, the IB MPXV model should be considered for further investigation of viral pathogenesis and countermeasures. To further our understanding of MPXV pathogenesis, we have expanded our studies to include molecular imaging applications to investigate factors that contribute to the survival of severe orthopoxvirus infection or that might be used for diagnosis or prognosis. One such factor, lymphadenopathy, is a distinctive feature of MPXV infection and was associated with disease severity in the recent United States outbreak. We have used positron emission tomography and computed tomography (PET/CT) with 18FDG as a nonspecific marker of metabolic activity to monitor inflammation and immune activation in NHPs. Groups of two NHPs were inoculated by the IV or IB route, and one animal survived in each group. Lymphadenopathy and increased immune activation represented by 18FDG uptake in the axillary lymph nodes (LNs) was evident in each NHP. Retrospective analysis of the axillary LNs indicated an association between pre-infection immune activation and survival. Both surviving IV- and IB-infected NHPs had significant 18FDG uptake in the LNs at the time of MPXV challenge with no clinical signs of illness, while the NHPs that succumbed did not. Massive inflammation in the lungs of IB-infected NHPs was also observed, and bilobular involvement was associated with moribundity. Molecular imaging was successfully used to monitor disease progression of an acute, severe viral infection and identify two patterns of immune activation/inflammation associated with survival. We have continued our study of cowpox (CPXV) infection as a BSL-2 model of orthopoxvirus infection and a surrogate model of variola virus infection. We have extended our studies in FY10 by demonstrating that IV inoculation with CPXV Brighton resulted in a uniformly lethal disease characterized by hemorrhagic manifestations. Surprisingly, the uniformly lethal dose for CPXV appears to be at least 100-fold lower than that observed for MPXV. Peak viremia levels reached greater than 8.0 log10genome copies/ml for NHPs infected with 5x10e5 PFU to 5x10e7 PFU CPXV. Lesion counts were less numerous than observed for MPXV with typically 50 or fewer lesions present in CPXV-infected NHPs. However, viral load in respiratory, lymphoid, and reticuloendothelial tissues was similar to levels observed in NHPs infected IV with MPXV. Investigations into countermeasures against the orthopoxviruses have also been pursued. We are currently evaluating broad-spectrum antiviral agents in collaboration with Functional Genetics, Inc. for in vitro and in vivo activity against the orthopoxviruses. In addition, a subset of host defense peptides (HDPs) with previously demonstrated immunomodulatory activities was selected for testing of direct or indirect antiviral function. In vitro antiviral activities were assessed using virus plaque assays or virus-GFP constructs through the following: 1) co-incubation of virus with HDPs, 2) pre-incubation of host cells with HDPs prior to infection, or 3) administration of HDPs to host cells post-infection. Two HDPs, as well as their protease-resistant all D-amino acid and retro-inverso analogs, demonstrated enhanced direct or indirect (immunomodulatory) antiviral activities against a broad range of viruses including cowpox, monkeypox, and vaccinia and were selected for testing in an intraperitoneal murine model of cowpox virus infection. Efficacy was demonstrated for both the immunomodulatory HDP and the all-D amino acid direct antiviral HDP with 40% survival in the lethal murine model following a single administration of peptide concurrent with infection. Currently these studies are being repeated in both intranasal and intraperitoneal models of cowpox infection and gene expression analysis will follow using the most efficacious infection model for determination of a putative mechanism of HDP action in vivo. 2. Countermeasures against viral hemorrhagic fevers. A safe and effective vaccine against Zaire ebola virus (ZEBOV) would potentially be used by multiple groups including healthcare providers, laboratory researchers, and military personnel. A vaccine could also be used to mitigate outbreaks in Africa and possibly be used to protect endemic nonhuman primate populations that have been affected by ZEBOV. While multiple strategies for ZEBOV vaccine development have been pursued, no current vaccine is close to being licensed by the Food and Drug Administration. Unresolved issues with current vaccine candidates include safety concerns, pre-existing immunity to vectored vaccines, and manufacturing and dosage obstacles. We are developing an alternative vaccine approach in collaboration with Dr. Matthias Schnell using attenuated rabies virus vaccine based vectors that express the major protective antigen of ZEBOV, the virion surface glycoprotein (GP). Rabies virus based vectors have previously been generated that express protective antigens from human immunodeficiency virus, hepatitis C virus, and anthrax protective antigen and found to be safe, attenuated, and efficacious in mice and/or nonhuman primates. The main objective of the present study is to determine the safety and level of immunogenicity of rabies virus vaccine-based vectors expressing ZEBOV GP as either live or killed vaccines and determine their efficacy in animal models of ZEBOV infection. Currently, we have generated an infectious cDNA clone of rabies vaccine virus expressing ZEBOV GP and recovered it in tissue culture. The recombinant viruses replicate to similar levels as the parental rabies vaccine strain and express ZEBOV GP in tissue culture. Analysis in mice for safety, immunogenicity, and protective efficacy is ongoing.